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517 Identification and characterization of selective MELK kinase inhibitors
168 Friday 21 November 2014 not by each inhibitor alone. Histomorphometric analysis showed that number of TRAP positive osteoclasts around tumor in bone was markedly decreased by TAS-115. In vitro study showed that HGF or VEGF produced phosphorylation of each receptors (MET or VEGFR2) -related signaling factors (AKT, ERK1/2) in murine osteoclasts, and TAS-115 suppressed them. These results suggested that although VEGFR inhibition is important for antitumor efficacy, the amelioration of tumor-associated bone disruption requires dual inhibition of MET and VEGFR. Conclusions: VEGFR and MET signaling cooperatively play pivotal roles for tumor-associated bone disruption, and TAS-115 is an effective therapeutic treatment for bone metastases. 516 POSTER Different genetic profiles of resistant and sensitive patients with EGFR wild type NSCLC undergoing tyrosine kinase inhibitor (TKI) treatment P.U. Ulivi1 , E.C. Chiadini1 , A.D. Dubini2 , D.C. Calistri1 , M.P. Puccetti3 , M.A.B. Burgio4 , A.D. Delmonte4 , A.V. Verlicchi5 , A.G. Gamboni6 , M.P. Papi7 , W.Z. Zoli1 , L.C. Crino` 8 , C.D. Dazzi5 . 1 IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.), Biosciences Laboratory, Meldola, Italy; 2 Morgagni-Pierantoni Hospital, Pathology Unit, Forl`ı, Italy; 3 S. Maria delle Croci Hospital, Pathology Unit, Ravenna, Italy; 4 IRCCS Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (I.R.S.T.), Medical Oncology, Meldola, Italy; 5 S. Maria delle Croci Hospital, Medical Oncology, Ravenna, Italy; 6 Degli Infermi Hospital, Medical Oncology, Faenza, Italy; 7 Infermi Hospital, Medical Oncology, Rimini, Italy; 8 Perugia Hospital, Medical Oncology, Perugia, Italy Background: Tyrosine kinase inhibitors (TKIs) are the first choice of treatment in a subset of patients with non-small cell lung cancer (NSCLC) harboring specific epidermal growth factor receptor (EGFR) mutations. However, about 15% of patients carrying wild type (wt) EGFR also respond to TKIs, and the reason for this sensitivity is unclear. Material and Methods: We considered a total of 60 patients with advanced NSCLC and EGFR wt who received erlotinib after one or more chemotherapy lines. Thirty patients were defined as responsive (stable disease or partial response for at least 6 months of treatment), while 30 were non-responsive. All patients had progressive disease at the start of treatment with erlotinib. Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor tissue or cytological tumor samples. A 26gene panel was analyzed by MassArray (Sequenom) using the LungCarta panel kit (Sequenom). Results: Sequenom analysis revealed that, among responsive patients previously identified as EGFR wt by pyrosequencing analysis, 2 (7%) had an EGFR mutation that was probably present in cell subclones and only detectable with more sensitive methods, while 7 (23%) showed a mutation in genes involved in the control of proliferation: 2 in PIK3CA, 1 in AKT , 1 in NTRK2 and 2 in STK11 genes. Moreover, 5 (17%) of the responding patients had a KRAS mutation (2 G12V,1 G12D, 1 G13D, 1 G12C). Of the 30 non responsive patients, 2 (7%) showed a resistance mutation in EGFR (P753S and L747S), 3 (10%) a KRAS mutation (G12D, G12C, G12V), 4 (13%) a p53 mutation (R273L, G245C, R158C, R273C) and 3 (10%) a NOTCH1 mutation (all R2328W). Conclusions: EGFR wt NSCLC patients showed different gene mutation profiles on the basis of their responsiveness or resistance to erlotinib. Responsive patients carried underrepresented EGFR mutations that were only detectable using highly sensitive methods and also harbored mutations in genes involved in different proliferation pathways. Conversely, EGFR resistance mutations, p53 and NOTCH 1 alterations were more frequently found in non-responsive patients. KRAS mutations were equally present in both groups of patients and would not seem to play a role in predicting response to TKIs. 517 POSTER Identification and characterization of selective MELK kinase inhibitors P. Carpinelli1 , N. Amboldi1 , D. Ballinari1 , S. Re Depaolini2 , U. Cucchi2 , G. Canevari3 , M. Caruso4 , R. Galli5 , D. Donati4 , A. Isacchi2 , E.R. Felder3 , A. Montagnoli1 , A. Galvani1 . 1 Nerviano Medical Sciences Srl, Cell Biology, Nerviano (Milano), Italy; 2 Nerviano Medical Sciences Srl, Biotechnology, Nerviano (Milano), Italy; 3 Nerviano Medical Sciences Srl, Chemical Core Technologies, Nerviano (Milano), Italy; 4 Nerviano Medical Sciences Srl, Medicinal Chemistry, Nerviano (Milano), Italy; 5 San Raffaele Scientific Institute, Neural Stem Cell Biology Unit, Milano, Italy MELK is a serine-threonine kinase that has been implicated in various cellular processes, including stem cell renewal, cell cycle progression, pre-mRNA splicing and apoptosis. MELK is overexpressed in various
Poster Session – Molecular Targeted Agents II undifferentiated tumors and its knockdown results in decreased cell-cycle progression, proliferation and tumor growth. Its expression has further been found to be enriched in embryonic and adult neural stem/progenitor cells and to be required for their self-renewal capacity. In tumor tissue, increased MELK expression correlates with the pathological grade of brain tumors, and is significantly correlated with poor prognosis of glioblastoma, breast and prostate cancer patients. Here we report the identification and biological evaluation of novel, selective inhibitors of MELK kinase. We identified a series of MELK inhibitors via high-throughput screening of our proprietary compound collection. Some of these small ATP-competitive molecules exhibit potent inhibitory activity against MELK enzyme with IC50 concentrations in the nanomolar range and are highly selective for MELK compared to a panel of protein kinases. Treatment of different tumoral cell lines with such compounds resulted in reduced cell proliferation with dose-dependent induction of apoptosis in a particular setting. Furthermore the compounds reduced neurosphere formation of glioblastoma stem cells, suggesting that they can have an effect on the growth of cancer stem cells. The threedimensional structure of a MELK fragment, comprising the kinase and UBA domains in complex with one of these compounds, NMS-P664, resolved at 2.5 A˚ resolution, reveals compound binding mode in the active site of MELK, thus complementing compound kinetic characterization and providing useful information for further optimization. Overall, our data demonstrated that the novel MELK inhibitors described above represent a valuable tool to further validate this kinase as a promising biological target and for the development of potential new anticancer therapies. 518 POSTER The HSP90 inhibitor, AT13387, overcomes resistance to crizotinib and second generation ALK inhibitors A. Courtin1 , A. Smyth1 , K. Hearn1 , V. Martins1 , J. Lewis1 , N. Thompson1 , J. Lyons1 , N. Wallis1 . 1 Astex Pharmaceuticals, Cambridge, United Kingdom Background: ALK-positive non-small cell lung cancer (NSCLC) can be successfully treated with the first generation ALK inhibitor, crizotinib, but responses are transient and tumors often relapse through a variety of resistance mechanisms. The second-generation ALK inhibitors can overcome some, but not all of these resistance mechanisms leaving a need for novel therapies to deal with resistance. The chaperone, HSP90, has many client proteins, including EML4-ALK, and HSP90 inhibition results in the disruption of multiple signalling pathways. Here we tested AT13387, an HSP90 inhibitor currently in Phase 2 clinical trials, in a number of preclinical models of crizotinib resistance to investigate its potential in ALK inhibitorresistant NSCLC. Materials and Methods: Crizotinib-resistant cell lines were generated by ex-vivo culture of H2228 xenograft tumors which had been treated continuously with crizotinib until relapse. Cell lines were characterized by proliferation assay and analysis of signalling by western blot. Results: Analysis of crizotinib-resistant cell lines suggested that different mechanisms were probably responsible for resistance. Two cell lines had increased levels of ALK whilst one cell line appeared to have lost ALK. One cell line had an activation of the STAT3 pathway. Signalling was investigated in these cell lines after treatment with the second generation ALK inhibitors, ceritinib and alectinib, and the HSP90 inhibitor, AT13387. As expected, signalling through the AKT and ERK pathways were poorly affected by crizotinib, but response varied to the 2nd generation ALK inhibitors with 2 cell lines being resistant to both alectinib and ceritinib as well as crizotinib. In contrast, treatment of all cell lines with the HSP90 inhibitor, AT13387, led to decreases in the levels of pERK, pAKT and pS6 suggesting that HSP90 inhibition can overcome resistance to these ALK inhibitors. All four compounds inhibited tumor growth of the ALK-positive H2228 xenograft model. AT13387 also inhibited tumor growth of an H2228-derived crizotinibresistant xenograft model. Conclusions: The HSP90 inhibitor, AT13387, is active in crizotinibresistant models and can overcome mechanisms of crizotinib resistance which are not susceptible to the second generation ALK inhibitors. This suggests AT13387 has therapeutic potential in ALK inhibitor-resistant NSCLC. AT13387 is currently being tested in the clinic in combination with crizotinib in ALK-positive NSCLC.
Poster Session – Molecular Targeted Agents II undifferentiated tumors and its knockdown results in decreased cell-cycle progression, proliferation and tumor growth. Its expression has further been found to be enriched in embryonic and adult neural stem/progenitor cells and to be required for their self-renewal capacity. In tumor tissue, increased MELK expression correlates with the pathological grade of brain tumors, and is significantly correlated with poor prognosis of glioblastoma, breast and prostate cancer patients. Here we report the identification and biological evaluation of novel, selective inhibitors of MELK kinase. We identified a series of MELK inhibitors via high-throughput screening of our proprietary compound collection. Some of these small ATP-competitive molecules exhibit potent inhibitory activity against MELK enzyme with IC50 concentrations in the nanomolar range and are highly selective for MELK compared to a panel of protein kinases. Treatment of different tumoral cell lines with such compounds resulted in reduced cell proliferation with dose-dependent induction of apoptosis in a particular setting. Furthermore the compounds reduced neurosphere formation of glioblastoma stem cells, suggesting that they can have an effect on the growth of cancer stem cells. The threedimensional structure of a MELK fragment, comprising the kinase and UBA domains in complex with one of these compounds, NMS-P664, resolved at 2.5 A˚ resolution, reveals compound binding mode in the active site of MELK, thus complementing compound kinetic characterization and providing useful information for further optimization. Overall, our data demonstrated that the novel MELK inhibitors described above represent a valuable tool to further validate this kinase as a promising biological target and for the development of potential new anticancer therapies. 518 POSTER The HSP90 inhibitor, AT13387, overcomes resistance to crizotinib and second generation ALK inhibitors A. Courtin1 , A. Smyth1 , K. Hearn1 , V. Martins1 , J. Lewis1 , N. Thompson1 , J. Lyons1 , N. Wallis1 . 1 Astex Pharmaceuticals, Cambridge, United Kingdom Background: ALK-positive non-small cell lung cancer (NSCLC) can be successfully treated with the first generation ALK inhibitor, crizotinib, but responses are transient and tumors often relapse through a variety of resistance mechanisms. The second-generation ALK inhibitors can overcome some, but not all of these resistance mechanisms leaving a need for novel therapies to deal with resistance. The chaperone, HSP90, has many client proteins, including EML4-ALK, and HSP90 inhibition results in the disruption of multiple signalling pathways. Here we tested AT13387, an HSP90 inhibitor currently in Phase 2 clinical trials, in a number of preclinical models of crizotinib resistance to investigate its potential in ALK inhibitorresistant NSCLC. Materials and Methods: Crizotinib-resistant cell lines were generated by ex-vivo culture of H2228 xenograft tumors which had been treated continuously with crizotinib until relapse. Cell lines were characterized by proliferation assay and analysis of signalling by western blot. Results: Analysis of crizotinib-resistant cell lines suggested that different mechanisms were probably responsible for resistance. Two cell lines had increased levels of ALK whilst one cell line appeared to have lost ALK. One cell line had an activation of the STAT3 pathway. Signalling was investigated in these cell lines after treatment with the second generation ALK inhibitors, ceritinib and alectinib, and the HSP90 inhibitor, AT13387. As expected, signalling through the AKT and ERK pathways were poorly affected by crizotinib, but response varied to the 2nd generation ALK inhibitors with 2 cell lines being resistant to both alectinib and ceritinib as well as crizotinib. In contrast, treatment of all cell lines with the HSP90 inhibitor, AT13387, led to decreases in the levels of pERK, pAKT and pS6 suggesting that HSP90 inhibition can overcome resistance to these ALK inhibitors. All four compounds inhibited tumor growth of the ALK-positive H2228 xenograft model. AT13387 also inhibited tumor growth of an H2228-derived crizotinibresistant xenograft model. Conclusions: The HSP90 inhibitor, AT13387, is active in crizotinibresistant models and can overcome mechanisms of crizotinib resistance which are not susceptible to the second generation ALK inhibitors. This suggests AT13387 has therapeutic potential in ALK inhibitor-resistant NSCLC. AT13387 is currently being tested in the clinic in combination with crizotinib in ALK-positive NSCLC.